Mitochondrial Ca(2+) signals in autophagy

Abstract

Macroautophagy (autophagy) is a lysosomal degradation pathway that is conserved from yeast to humans that plays an important role in recycling cellular constituents in all cells. A number of protein complexes and signaling pathways impinge on the regulation of autophagy, with the mammalian target of rapamycin (mTOR) as the central player in the canonical pathway. Cytoplasmic Ca(2+) signaling also regulates autophagy, with both activating and inhibitory effects, mediated by the canonical as well as non-canonical pathways. Here we review this regulation, with a focus on the role of an mTOR-independent pathway that involves the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel and Ca(2+) signaling to mitochondria. Constitutive InsP(3)R Ca(2+) transfer to mitochondria is required for autophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to insufficient production of reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer to mitochondria results in activation of AMPK, which activates mTOR-independent pro-survival autophagy. Constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration, maintenance of normal cell bioenergetics and suppression of autophagy.

Fig. 1

A mechanism for activation of autophagy by elevated cytoplasmic [Ca²⁺] in the canonical mTOR pathway. Various Ca²⁺ mobilizing agents have been shown to induce autophagy. Activation of calmodulin (CaM) by Ca²⁺ activates CaMKKβ, which phosphorylates and activates AMPK. Activated AMPK phosphorylates the TSC1/TSC1 complex, enhancing its GTPase activity to maintain Rheb in its GDP-bound inhibited state. Absence of Rheb-GTP activity inactivates mTORC1, releasing its break on autophagy. Inhibition of elevated [Ca²⁺]_i by buffering it with BAPTA prevents activation of CaMKKβ and AMPK, enabling Rheb to maintain mTORC1 activity, suppressing autophagy.

Fig. 2

Suppression of InsP₃R Ca²⁺ transfer to mitochondria induces mTOR-independent autophagy. A. In resting cells, low-level InsP₃ production is maintained by circulating ligands for receptors that couple to phospholipases C (PLC) beta and gamma. In addition, InsP₃R-mediated Ca²⁺ release and/or extracellular influx the Ca²⁺ can activate calpain, which cleaves and activates the heterotrimeric G-protein alpha subunit G_s, increasing the levels of cAMP, activating Epac to stimulate the small G protein Rap2B that activates PLCε. Phosphatidylinositol 4–5-biphosphate (PIP₂) levels are maintained by a constant supply of inositol (Ins) by the inositol monophosphatase (IMPase). Low-level stochastic Ca²⁺ release from the endoplasmic reticulum by InsP₃R, enhanced by Bcl-2 and Bcl-x_L bound to the channel, provides Ca²⁺ to mitochondria in close proximity, mediated by the outer membrane VDAC and MCU at the inner mitochondrial membrane. Matrix Ca²⁺ activates pathways, including dehydrogenases and the F₁-F₀-ATPase, that fuel the electron transport chain and O₂ consumption by providing reducing equivalents in the form of NADH. ATP production maintains a low cytoplasmic AMP:ATP ratio. This mechanism provides the cell with bioenergetic fitness that suppresses autophagy. B. The absence of constitutive delivery of Ca²⁺ from ER to mitochondria, due either to inhibition of InsP₃R-mediated Ca²⁺ release (by genetic deletion of InsP₃R (DT40-KO), inhibition of InsP₃R activity (by xestospongin B (XeB), molecular ablation of the InsP₃R (by RNAi), inhibition of InsP₃ production by blocking the IMPase (by lithium (Li⁺) or L-690,330), blocking PLC (not shown; [37]), or inhibiting the calpain cascade that impinges on PLCε)); or by inhibition of mitochondrial Ca²⁺ uptake (Ru360 or MCU RNAi), results in diminished pyruvate dehydrogenase (PDH) activity (as well as the activities possibly other Ca²⁺-sensitive dehydrogenases and the ATP-synthase) that results in insufficient production of NADH, limiting the activity of the electron transport chain, diminishing O₂ consumption and reducing ATP production. The consequent rise of the AMP:ATP ratio activates AMPK, which induces pro-survival, mTOR-independent autophagy, possibly involving ULK1/2 and/or sirtuin1.

Fig. 2

Suppression of InsP₃R Ca²⁺ transfer to mitochondria induces mTOR-independent autophagy. A. In resting cells, low-level InsP₃ production is maintained by circulating ligands for receptors that couple to phospholipases C (PLC) beta and gamma. In addition, InsP₃R-mediated Ca²⁺ release and/or extracellular influx the Ca²⁺ can activate calpain, which cleaves and activates the heterotrimeric G-protein alpha subunit G_s, increasing the levels of cAMP, activating Epac to stimulate the small G protein Rap2B that activates PLCε. Phosphatidylinositol 4–5-biphosphate (PIP₂) levels are maintained by a constant supply of inositol (Ins) by the inositol monophosphatase (IMPase). Low-level stochastic Ca²⁺ release from the endoplasmic reticulum by InsP₃R, enhanced by Bcl-2 and Bcl-x_L bound to the channel, provides Ca²⁺ to mitochondria in close proximity, mediated by the outer membrane VDAC and MCU at the inner mitochondrial membrane. Matrix Ca²⁺ activates pathways, including dehydrogenases and the F₁-F₀-ATPase, that fuel the electron transport chain and O₂ consumption by providing reducing equivalents in the form of NADH. ATP production maintains a low cytoplasmic AMP:ATP ratio. This mechanism provides the cell with bioenergetic fitness that suppresses autophagy. B. The absence of constitutive delivery of Ca²⁺ from ER to mitochondria, due either to inhibition of InsP₃R-mediated Ca²⁺ release (by genetic deletion of InsP₃R (DT40-KO), inhibition of InsP₃R activity (by xestospongin B (XeB), molecular ablation of the InsP₃R (by RNAi), inhibition of InsP₃ production by blocking the IMPase (by lithium (Li⁺) or L-690,330), blocking PLC (not shown; [37]), or inhibiting the calpain cascade that impinges on PLCε)); or by inhibition of mitochondrial Ca²⁺ uptake (Ru360 or MCU RNAi), results in diminished pyruvate dehydrogenase (PDH) activity (as well as the activities possibly other Ca²⁺-sensitive dehydrogenases and the ATP-synthase) that results in insufficient production of NADH, limiting the activity of the electron transport chain, diminishing O₂ consumption and reducing ATP production. The consequent rise of the AMP:ATP ratio activates AMPK, which induces pro-survival, mTOR-independent autophagy, possibly involving ULK1/2 and/or sirtuin1.